ABSTRACT There is an urgent need to improve the efficacy of chemoradiation therapy for patients with locally advanced pancreatic cancer (LAPC). Since unrepaired DNA double-strand breaks (DSB) are responsible for the majority of cell death in response to chemoradiation, targeting cellular DNA damage response (DDR) pathways is a promising approach to enhance the efficacy of chemoradiation therapy by preventing efficient repair of DNA damage, leading to loss of tumor cell viability. Since defects in the DDR such as P53 mutation occur in up to 70% of pancreatic ductal adenocarcinomas (PDAC), targeted inhibition of the DDR provides an opportunity to selectively enhance sensitivity to chemoradiation in tumor but not normal cells. The overall goal of our proposed study is to identify agents from the CTEP collection that modulate the cellular response to DNA damage and best sensitize tumor cells to standard of care chemoradiation therapy. To achieve this goal, we will in Aim 1 conduct an unbiased high throughput screen (HTS) in a panel of PDAC cell lines using a panel of agents from the CTEP collection that modulate the cellular DDR, including drugs that target ATR, DNAPK, PARP, and WEE1 (Aim 1A). An initial cell viability screen at various concentrations of single agents alone or in combination with chemoradiation will be conducted. In addition to concentration, optimal sequencing of targeted agents with chemoradiation will be investigated. A live cell assay for the DDR as well as apoptotic cell death will be utilized as orthogonal screens to assist in hit prioritization and validation. In Aim 1B, a group of agents selected by defined criteria will be further evaluated by conducting a ?gold standard? clonogenic survival assay using a panel of PDAC cell lines. In Aim 1C we will conduct a normal cell counter screen using the above agents against normal cells to identify those which cause toxicity in combination with chemoradiation. The efficacy of these prioritized agents will be validated in Aim 1D using a panel of patient- derived xenograft (PDX) sphere explant cultures. Two agents with the optimal enhancement ratio (1B and 1D), and safety (1C) at the optimal concentration and schedule (1A and 1B) will be studied in Aim 2. We will compare the therapeutic efficacy of the nominated agents in combination with chemoradiation using both PDX and immune competent syngeneic mouse models of PDAC (Aim 2A). Normal tissue injury caused by these two agents (Aim 2B) will be evaluated, which together with tumor efficacy will lead to the choice of the agent to pursue in clinical trials. Aim 3 will delineate the mechanistic basis for the chemoradiation potentiating activity of the superior agent (Aim 3A) with the goal of selecting pharmacodynamic and imaging biomarkers (Aims 3B and 3C), using in vivo studies in mouse models, for the future clinical trial. Completion of these aims will provide the required preclinical data regarding efficacy, safety, and biomarkers of response (mechanistic- and imaging- based) for the rationale design of a future clinical trial for patients with LAPC.